EP0712936B1 - Niedermolekulare dna aus störmilch, methode zur gewinnung der dna und ein pharmazeutisches präparat darauf basierend - Google Patents

Niedermolekulare dna aus störmilch, methode zur gewinnung der dna und ein pharmazeutisches präparat darauf basierend Download PDF

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EP0712936B1
EP0712936B1 EP94913856A EP94913856A EP0712936B1 EP 0712936 B1 EP0712936 B1 EP 0712936B1 EP 94913856 A EP94913856 A EP 94913856A EP 94913856 A EP94913856 A EP 94913856A EP 0712936 B1 EP0712936 B1 EP 0712936B1
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Prior art keywords
dna
preparation
pharmaceutical preparation
day
low
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EP94913856A
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English (en)
French (fr)
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EP0712936A1 (de
EP0712936A4 (de
Inventor
Alexandr Vilenovich Asafov
Vyacheslav Vladimirovich Bezjulev
India Grigorievna Anosova
Alexandr Andreevich Konarev
Igor Nikolaevich Karpov
Marina Gavrilovna Kochetkova
Ivan Dmitrievich Treschalin
Dmitry Alexandrovich Bodyagin
Elena Mikhailovna Treschalina
Eleonora Rafailovna Pereverzeva
Anatoly Borisovich Syrkin
Viktor Vladimirovich Ilyashenko
Natalya Alexandrovna Susuleva
Lev Abramovich Durnov
Olga Borisovna Zobova
Irina Lazarevna Kazarova
Nelli Borisovna Koroleva
Stepan Vazgenovich Kirakosian
Lidia Vasilievna Simonova
Larisa Alexandrovna Tarasova
Ljudmila Vasilievna Simonova
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OBSCHESTVO S OGRANICHENNOI OTVETSTVENNOSTIJU "NAUC
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NAUCHNO-PROIZVODSTVENNOE PREDPRIYATIE "FARMEK"
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Priority claimed from RU93020154A external-priority patent/RU2043767C1/ru
Priority claimed from RU93020155/14A external-priority patent/RU2034554C1/ru
Priority claimed from RU93026472/14A external-priority patent/RU2049461C1/ru
Priority claimed from RU93046328/14A external-priority patent/RU2094438C1/ru
Priority claimed from RU93046329/14A external-priority patent/RU2078581C1/ru
Priority claimed from RU93046326A external-priority patent/RU2071780C1/ru
Priority claimed from RU93046327/14A external-priority patent/RU93046327A/ru
Application filed by NAUCHNO-PROIZVODSTVENNOE PREDPRIYATIE "FARMEK" filed Critical NAUCHNO-PROIZVODSTVENNOE PREDPRIYATIE "FARMEK"
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Publication of EP0712936A4 publication Critical patent/EP0712936A4/de
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations

Definitions

  • the present invention relates to biotechnology, medicine and cosmetology and concerns the low-molecular DNA extracted from milt of sturgeons, a new preparation on its base and a method for extraction of said DNA.
  • the preparation according to the invention possesses a wide range of biological activity and can be utilized in the capacity of a biocorrector, inhibitor of viral activity, HIV (human immunodeficiency virus) infection for one, and lesions of the mucous membrane of various etiology.
  • the preparation will also be useful as a universal cosmetic addition in lotions, creams, dentifrice water, etc.
  • cytostatic therapy the backbone of medicamentous treatment of tumors, often leads to inhibition of hemopoiesis, even in therapeutic doses (1).
  • the toxic myelodepression is registered in peripheral blood as leukopenia, thrombocytopenia and, further, as anemia (2).
  • Myelosuppression concomitant with chemo- and chemoradiation therapy limits the treatment possibilities, raises the risk of bacterial infections and lethal termination (1, 2).
  • the range of means used in case of hematogenesis depression is sufficiently wide (3,4). It includes transplantation of bone marrow, introduction of leukomass, colony-stimulating factors, and such preparations as splenin, leukogene, pentoxyl, eimosane and various groups of vitamins (5-7).
  • the hemocorrector injected in cases of leukopenia and agranulocytosis was sodium salt of ribonucleic acid produced by hydrolysis from yeast (9).
  • the preparation has the same disadvantages, i.e. it stimulates leukopoiesis only, is low effective and calls for cancellation of the course of chemotherapy.
  • the sodium salt of low-molecular DNA produced from milt of sturgeons and not used heretofore features unexpected biological activities such as effective stimulation of leukopoiesis, including bone marrow hematogenesis, stimulation of healing the lesions of mucous membranes of various localization and etiologies, inhibition of viral and microbial activities, particularly HIV infection.
  • the HIV infection was suppressed by nucleotide analogs, e.g. AZT (3-azi-3-desoxythymidine) (12, 13).
  • nucleotide analogs e.g. AZT (3-azi-3-desoxythymidine) (12, 13).
  • this preparation is effective only at early stages of the disease and, even in therapeutic doses, is highly toxic which leads in clinical use to complications (headache, anemia, disorders of gastroenteric tract) and thus restricts its administration (14,15).
  • Known in the prior art of treating HIV infection is the administration of difluorinated nucleosides (16) which, however, are little effective.
  • the claimed preparation features a high efficiency with respect to inhibition of HIV infection, and a low toxicity.
  • the preparation based on low-molecular DNA obtained from milt of sturgeons has demonstrated an unexpectedly high efficiency with respect to lesions of oral and pharyngeal mucous membrane of various etiology including those resulting from administration of antibiotics, preparations of heavy metals, and cytostatics.
  • a method for producing DNA from milt of sturgeons has been disclosed in Inventor's Certificate USSR No. 1410494, 1986, C074221 (04).
  • the method involves homogenization of milt in a standard citrate-salt buffer, separation of cell nuclei by centrifuging, homogenization and deproteinization with a solution of dodecyl sulfate Na at a temperature of 55 C approximately.
  • the cooled mixture is vigorously mixed with kieselguhr (15.5 %) and filtered.
  • the target DNA is precipitated with alcohol in a 1:1 proportion.
  • the high-polymeric DNA is dissolved and subjected to the effect of sound on a magnetostrictor, and filtered, producing 0.6 % DNA in 1/10 standard citrate-salt buffer.
  • Also known in the prior art is a method for production of DNA from sturgeon milt involving homogenizing of milt in a standard citrate-salt buffer (0.87% Na Cl, 0.54 sodium citrate), centrifuging, deproteinizing in presence of dodecyl sulfate Na in the course of 1.5 h at 60 C.
  • the suspension is treated with NaCl and citrate Na to final concentrations of 14.6 % and 24 %, respectively, and heated for 1.5 h at 60 C.
  • the cooled solution is passed through a column with kieselguhr, producing a DNA solution (Chemo-Pharmaceutical Journal, 1982, No. 7, p. 835-838).
  • the present invention relates to a compound of the low-molecular DNA produced from sturgeon milt, to a pharmaceutical preparation containing such a compound, and to a method for extraction of low-molecular DNA from sturgeon milt, as claimed in attached claims 1, 4 and 16 respectively.
  • the sturgeon milt is homogenized in a standard citrate-salt buffer solution composed of 0.87 % sodium chloride and 0.54 % sodium citrate.
  • the homogenate is heated at 55-60 C for 1.5 h in presence of sodium dodecyl sulfate (final concentration 96 %).
  • the mixture is treated with Na Cl solution to a final concentration of 14.6 % and heated for 1.5 h at 55-60 C.
  • the produced mixture is treated with kieselguhr and filtered.
  • the resulting solution of DNA is passed through an electrodializer at a temperature of 15-20°C and a current density of 1.0-1.5 A/sq dm and/or ultrafiltration plant at a temperature of 20-24°C and a pressure of 2-3 kg/sq cm.
  • the obtained solution is treated by ultrasound, filtered, concentrated if necessary, precipitated with alcohol and the precipitate is washed with 96 % alcohol, dried in a drying cabinet at 40 C, producing DNA in dry powder form.
  • the salt of DNA obtained by the above-described process features the following properties.
  • the molecular weight of sodium salt of DNA is not over 12 kD and its hyperchromic effect, not under 37%.
  • the product prepared in accordance with the claimed method is suitable for medical utilization (market name Polydan).
  • the preparation according to the invention can be used in different forms for different purposes.
  • the solution of sodium salt of DNA is used in the form of injections.
  • the diluent may be any pharmaceutically-acceptable diluent including distilled water and physiological solution.
  • the preparation for injection contains DNA in the amount required for exhibiting its hemostimulating effect.
  • the preferable content of DNA in the preparation for injections varies from 1% to 2% since this concentration ensures an optimum pharmacological effect.
  • the claimed preparation is made in factory conditions by a traditional volumetric-weight method (17). A certain weight amount of DNA powder is treated with diluent to the required volume of the solution. Then the solution is filtered, sterilized and poured out into vials.
  • the diluent is preferably distilled water or physiological solution.
  • the resultant solution is a transparent liquid, contains no mechanical or other impurities. It is stable and characterized by long shelf life.
  • an efficient solution contains DNA in amounts sufficient for healing them.
  • the preferable DNA concentration in the solution is 0.2 to 2.0 %.
  • the diluent may be, apart from physiological solution and water,any other pharmaceutically-acceptable diluent.
  • DNA is used in combination with polyvalent metals (zinc, nickel, cobalt, iron) in 1:1 to 1:1000 proportions.
  • the metal systems are produced by mixing components in certain proportions and at different temperatures.
  • the DNA according to the invention used as a general-purpose biological addition in cosmetics can be utilized in various forms.
  • the foundation of face-cream contains glycerine stearate, distilled monoglycerides or adequate substitutes thereof, emulsion wax, cosmetic stearin, triethanolamine, glycerine, perfumery oil, conservants, aromatizer, and water.
  • Hair lotions are based on ethyl alcohol, solubilizer, gelling components, glycerine, 1,2-propyleneglycol, aromatizer, and water.
  • Lotions are prepared by mixing the gelling components with water then adding the remaining components.
  • Cream is prepared by a traditional method (18), i.e. by mixing fatty and water phases. DNA is added to the cream mass under certain conditions (temperature, pH, adding sequence).
  • dentifrice water The basic components of dentifrice water are ethyl alcohol, distilled glycerine, aromatizer, food dyestuffs.
  • the dentifrice water is prepared by the traditional method (18,19), i.e. by mixing components.
  • the concentration of the preparation was 0.1; 1.0; 10; 100 and 1000 mkg/ml.
  • the solution of the studied preparation was prepared directly before administration.
  • the preparation was injected subcutaneously in 0.2 ml of NaCl solution in a dose of 5 mg/kg of animal's body weight (5 toxic doses) and in 100 mg/kg of body weight (100 toxic doses).
  • the control group was injected in the same way with the same volume of diluent. Identical doses were used in the 3rd series of experiments.
  • the preparation in the ready-made form (1.5 % solution) was administered to animals subcutaneously or intravenously one or five times with 24-h intervals depending on the purpose and objects of the experiment. Tests were made on male rats F 1 (SVA x C 57 VC) and SVA, male and female rats Wistar and F (AMSY and Wistar), Guinea pigs, English beagle dogs and chinchilla rabbits. The condition and behavior of animals were observed within a month after the end of the course of administering the preparation.
  • the toxicity doses were calculated by the Litgfield and Wilkokson method and after a month the animals were killed and subjected to postmortem examination.
  • mice F 1 SVA x C 57 VC weighing 18-20 g in doses of 1000, 500 and 250 mg/kg.
  • Intravenous injection of the preparation to the mice of the same line was made in the doses of 600, 300 and 150 mg/kg (the volumes were, respectively, 0.3, 0.15 and 0.07 ml per mouse).
  • the toxicity of DNA complexes with the above-mentioned polyvalent metals was studied on nonlinear mice weighing 6-7 g by making subcutaneous, intraperitoneal and intracerebral injections in 5, 10, 20, 35 and 50, 75 mg/kg doses. The animals were observed for two weeks after which LD 50 was calculated.
  • mice F 1 male mice F 1 (AMSU x Wistar) weighing 220-250 g on an average.
  • the pathomorphological examination was conducted by killing 5 animals from each group by decapitation on the 14th and 30th day after the completion of the course. Then the animals were dissected, their internal organs weighed and their weight coefficients determined. Pieces of organs were fixed in a 10-% solution of formalin, colored with hematoxylin-eosine and subjected to optical microscopy.
  • the examination has shown that the preparation is easily endured by the animals. There were no abnormalities in the behavior and condition of the animals. Administration of the preparation was not accompanied by a pain syndrome.
  • the hemocorrecting properties of the preparation have been disclosed on animals (mice, dogs) under the conditions of experimental leukocytopenia caused by cytostatics.
  • the content of leukocytes and karyocytes in control animals remained low up to the 28th day of the experiment and began to be slowly restored after the 42th day but failed to reach the initial level to the end of observation, on the 49th day.
  • the second series of experiments was carried out on male hybrid mice F 1 (CBAxS 57 BS) weighing 18-20 g. Cytopenia was caused by administering cyclophosphane intraperitoneally in single doses of 200 and 275 mg/kg. The sodium salt of DNA was injected subcutaneously by single shots in doses of 150 and 30 mg/kg and on the 3rd, 5th and 7th days after cyclophosphane injection. On the 1st, 3rd, 5th, 7th, 10th, 12th, 14th, 17th, 21st and 25th days the total number of leukocytes in peripheric blood was determined in the Goryaev chamber. The results of the experiment are summarized in Table 4.
  • DNA does not cause any compensating reduction of cell number after hemostimulation.
  • RNA yeast As distinct from the known preparation containing yeast RNA, administration of the claimed preparation speeds up the recovery of the leukocyte number in peripheric blood and increases the number of karyocytes in the bone marrow.
  • One injection of DNA preparation is sufficient for producing the stimulating effect while the use of RNA yeast achieves the desired effect only after a 10-days treatment.
  • the claimed preparation does not cause compensating reduction of cell number after hemostimulation.
  • the clinical investigations of the claimed preparation have been carried out in oncological patients in connection with inhibition of hemopoiesis caused by chemoradiation therapy.
  • the observed group consisted of 30 patients within a 6-14-year age bracket suffering from lymphosarcoma, lymphoblastic leucosis, etc.
  • Treatment with cytostatics and radiation caused the development of leukopenia (100 to 1500 per cu.cm of blood).
  • the course of treatment included the preferable use of 0.5-2.5-% solution of low-molecular DNA produced from the milt of sturgeons.
  • the doses and number of injections were varied in accordance with the course of the main ailment.
  • the most preferable was the dose interval from 7 to 35 mg/kg ensuring a 3-5-fold increase of leukocytes after 1 or 2 injections.
  • the claimed preparation exhibits not only a leukostimulating effect by far more intensive than that of the previously known preparation but also an ability unknown heretofore to stimulate the bone marrow hemogenesis.
  • Sodium nucleinate Na sodium salt of nucleic acid produced by hydrolysis of yeast is used as a hemocorrector in cases of leukopenia.
  • the factor of stimulating the colonies of granulocytes and macrophages is the most frequently used hemocorrector in leukopenias resulting from chemo-radiotherapy.
  • the HIV infection can be inhibited, according to the present invention, by the sodium salt of DNA produced from sturgeon milt in combination with polyvalent metals (zinc, nickel, cobalt, iron) in a ratio from 1:1 to 1:1000.
  • the preparation can be administered by subcutaneous, intraperitoneal and intranasal methods or into the cerebrospinal canal, depending on the form of the disease, in therapeutically-equivalent doses.
  • the antiviral effect achieved in different forms of administering the preparation is vital for various clinical forms of AIDS-pulmonary and gastroenteric with damage to the central nervous system. (14).
  • High efficiency of the preparation according to the invention in inhibiting the HIV infection was discovered in the following experiments.
  • HIV in the cell culture systems was inhibited by the DNA complexes in combination with zinc, cobalt, nickel and iron at various concentrations of components.
  • Azidothymidine manufactured by the Welcome Co. was used for comparison.
  • the antiviral activity of preparations was assessed by the methods recommended for this purpose by the World Health Organization (20, 21).
  • the cells were cultured at a concentration of (0.03-0.05)x10 6 cells per 1 ml of PPM1 1640 with 10-% fetal serum of calves, 300 mg/ml of glutamine, 100 mkg/ml of heptamicine and grown in the form of a suspension.
  • the vital activity of cells was checked by coloring them with a 0.4-% solution of trypan blue.
  • the virus sources were strains HIV/IVS and HIV - 1 HTLV/IIIB.
  • the suspension of cells was placed into 24-hole boards, treated with different doses of the preparation and infected with HIV.
  • the multiplicity of infection was 0.01 TCD 50 per cell.
  • the cultures were incubated at 37 C in the atmosphere containing 5% CO 2 at a moisture content of 98% for 5-7 days before determining the cytopathic effect of the virus on the cell culture.
  • Tests were made concurrently with all four types of the preparation in comparison with azidothymidine in 5, 10, 20, 35 and 50 mg/kg doses. Investigations of toxicity of the four types of the preparation and azidothymidine were conducted on nonlinear white mice weighing 6-7 g with preparations administered in different doses in the form of subcutaneous, intranasal, intraperitoneal and intracerebral injections. The animals were kept under observation for two weeks and calculations were made by the Curber method. The LD 50 was determined as a dose of the preparation causing death of 50% of animals.
  • the antiviral activity of DNA-metal complexes was also investigated with respect to the influenza virus, adenoviruses and herpes viruses, using the conventional methods (Mc. Canley J., Biochem.J. 1983, v. 224, p. 281; Wigand R. et al, Entevirology, 1982, v. 18, p. 169; Allen G.P., Aust.J. Vet. Res. 1983, v. 44, p. 263).
  • the obtained data are presented in Table 11.
  • the efficiency of nucleic acids in healing the lesions of mouth and pharynx mucous membranes has been unknown heretofore.
  • the claimed preparation exhibits a possibility of stimulating the healing of lesions of the mouth and pharynx mucous membranes of various etiologies.
  • Most crucial today is the problem of healing the mucous membrane lesions of the oral and pharynx cavity which are frequent complications after local and parenteral administration of antibiotics, preparations of heavy metals, and cytostatics (22-24).
  • chemotherapeutic preparations such as methatrexate, vincrysgin, prednisolon, etc.
  • the Table shows that as early as on the 2nd day of treatment the pains vanished, food could be taken on the 2nd-3rd days and solid food, on the 4th day.
  • the preparation according to the invention exhibits an efficient cosmetic effect. It must be borne in mind that the DNA has not been used heretofore in the capacity of a cosmetic biologically-active addition.
  • Said cream was found to activate the proliferative processes in the skin without any irritating and allergenic effect.
  • Toxicity was investigated by the Gatsuro method (27). The irritating effect was assessed by applying the preparation to the mucous membrane of the rabbit's eye. The tested lotion was also applied daily for 21 days to an area of skin 2x2 cm in size.
  • the effect of the lotion on the linear growth of hair was studied on Guinea pigs.
  • the animal's hair was sheared from a certain area and said area was treated for 21 days with 0.5 g of lotion.
  • the hair was torn out and its length was measured.
  • the structure of hair was studied and the results were compared with the control.
  • Toxicity of the preparation was studied for 21 days.
  • the general condition, appetite, weight gain, condition of skin, hair and presence of edemas were also observed. Bioptates of skin and internal organs were examined visually and pathomorphologically.
  • the lotion was rubbed in daily for 21 days. Before the course of treatment, flap samples were made for 24 h under a closed bandage.
  • the preparation features good organoleptic properties, can easily be applied to the hair-covered part and is devoid of an allergenic effect.
  • the sodium salt of DNA according to the invention imparts an antiinflammatory, antiseptic and antiparodontosis activity to the dentifrice water.
  • DNA according to the invention is a universal addition to any cosmetic products, features pronounced curative and prophylactic properties without causing any allergenic and other side effects, does not affect adversely the normal structure of the skin and internal organs and is compatible with the active principle of any cosmetic product.
  • the patient K. admitted to hospital in connection with myelodepression caused by chemo-radiotherapy was administered 5 ml of 1.5 % solution of the sodium salt of DNA.
  • leukopenia was 100-300 leukocytes per cu mm of blood, but after administration a gradual increase in the number of leukocytes was observed.
  • the maturing index of erythrokaryocytes before treatment with the claimed preparation was equal to zero, while after treatment it was 0.8 (normal value being 0.7-0.9).
  • the patient A., 13 was admitted into the department of infantile oncology on the 13th of July 13, 1992.
  • the diagnosis was acute lymphoblastic leukosis.
  • a course of chemotherapy with cyclophosphane, vinkristin, rubomicine and prednisolon was administered.
  • the result was increasing leukopenia.
  • the number of leukocytes was 2500, on November the 27th, 1992, 400, on November the 28th, 1992, 300, and on November the 29th, 100.
  • the patient was injected with 5 ml of 1.5% solution of polydan. The result was an increase in the number of leukocytes.
  • the number of leukocytes was 700, on December the 7th, 4200, on December the 10th, 5000.
  • the myelogram was normalized: the number of myelocytes, erythrokaryocytes, polychromatophylic normocytes increased which was an evidence of the stimulation of bone marrow hemogenesis.
  • a culture of inoculated cells, line CEMSS and MT-4 was used in the experiment.
  • the cells were cultivated at a concentration of 0.03x0.05x10 6 cells per 1 ml of PPMJ 1640 medium with 10-% fetal serum of calves, 300 mg/ml of L glutamin, 100 mkg/ml of hentamycine and were grown in the form of a suspension.
  • the vital activity of the cells was checked by coloring them with a 0.4-% solution of trypan blue.
  • Strains HIV/IVS and HIV-LHTLV/IIIB were used for viral sources.
  • the suspension of cells was placed into 24-hole plates, treated with various doses of DNA-Zn complex at a DNA-metal relation of 1:1, 4:9, 3:10, 0.5:350, 1:1000 and infected with HIV.
  • the multiplicity of infection was 0.01 TCD 50 per cell.
  • the cultures were incubated at 37 C in the atmosphere containing 5% of CO at a moisture content of 98% for 5-7 days before determining the cytopathic effect of the virus on the cell culture.
  • the formation of viral antigen was assessed by an immunofermental analysis.
  • the experiment has shown that the number of syncytia, per cent of virus control at a DNA-metal relation of 3:10 was 25% at 69% of viable cells; at a relation of 0.5:350, 35% at 81.2 % of viable cells; at a relation of 1:1000, 70% and 84.3 % of viable cells; and at a relation of 1:1100, 100% and 86.2 % of viable cells.
  • the suspension of cells was processed with various doses of DNA-Ni complex at the same relations of components as in Example 1.
  • the experiment has shown that the number of syncytia, per cent of virus control at a DNA:Ni relation of 3:10, 0.5:350 was zero at a viability of cells of 81 and 90%; at a relation of 1:1000, it was 68% at 84.3 % of viable cells and at a relation of 1:1100, it was 100% at 87,5 % of viable cells.
  • the suspension of cells was treated with various doses of DNA-Co complex at the same relations of components as in Example 1.
  • the experiment has shown that the number of syncytia, per cent of virus control at a DNA-Co relation of 3:10 is 10% at 78.4 % of viable cells; at a relation of 0.5:350 it was 15% at 87.1 % of viable cells; at a relation of 1:1000 it was- 66% at 76.3 % of viable cells, and at a relation of 1:1100 the viability was, respectively, 82.1 %.
  • the suspension of cells was treated with various doses of DNA-Fe complex at the same relations of components as in Example 1.
  • the experiment has shown that the number of syncytia, per cent of virus control, at DNA-Fe relations of 3:10 and 0.5:350 was zero at a viability of cells of 79.3 and 82.1 %, respectively.
  • the number of syncytia was 61% at a cell viability of 81.7 %; and at a relation of 1:1100 it was 100% at a cell viability of 84.5 %.
  • the patient P. was admitted with the main diagnosis of sarcoma, IV degree. Was given metatrexate in a dose of 50 ml/sq m.
  • the symptoms of stomatitis developed: the oral cavity turned hyperemic with edemas and aphthous eruptions, sharply painful. Taking of food was impossible.
  • the patient was administered applications of 0.25- % solution of polydan on the first day, eight times with 2-h intervals; on the 2nd-6th days, eight times daily; six times on the 7th day and three times on the 8th day.
  • the dynamics of clinical symptoms of stomatitis was the following: on the 2nd day after treating the mucous membrane the pains vanished, the aphthous symptoms stabilized, bleeding stopped. On the 3rd-4th day the pains vanished, the aphthous elements diminished, the patient started taking food; on the 5th day there were no pains, hyperemia vanished; on the 6th day the process was fully arrested, the mucous membrane of the oral cavity was clean, there were no edemas and hyperemia.
  • the stomatitis was treated without cancelling chemotherapy.
  • the patient K. was admitted with the main diagnosis of lymphosarcoma, IV degree. was administered 500 ml/sq m of metatrexate and 2 mg/kg of prednisolon.
  • the patient was administered applications of 0.5-% solution of polydan: eight times a day (every 2-3 h) on the lst-4th day; six times a day on the 5th-8th days.
  • the dynamics of the clinical symptoms of gingivostomatitis was the following: abatement of pains on the 2nd day after treatment of the mucous membrane; on the 3rd day - vanishing of pains, stopped bleeding, taking of food possible; on the 4th-5th day the hyperemia of the mucous membrane diminished and on the 8th day the process was completely arrested. On the 9th day the mucous membrane of the oral cavity and gums was clean, of pale-pink color, without any signs of edema and hyperemia. The gingivostomatitis was treated without cancelling of polychemotherapy.
  • the patient K. was admitted with the main diagnosis of acute leucosis and treated with 500 mg/sq.m of metatrexate.
  • the dynamics of medicamentous pharingostomatitis was as follows: on the 3rd day painfulness receded, the patient began taking food, on the 4th day the bleeding and number of aphthae diminished, on the 5th-6th day there remained but a small number of eruptions, on the 7th-9th days hyperemia remained. On the 10th day the process was completely stopped. The mucous membrane of the oral and pharyngeal cavity was found to be clean, of a pink color, free of edemas. Esophagostomatitis was treated without cancelling chemotherapy.
  • the above examples confirm the efficiency of the polydan solution for treating medicamentous lesions of the mucous membrane of the mouth and pharynx.
  • aqueous phase 0.1 (w%) of triethanolamine, 2.0 glycerine and water are heated to 75-80 C.
  • the fatty phase is prepared by heating 0.5 lanolin, 1.0 distilled monoglycerides, 0.5 cosmetic stearine, 2.0 perfumery oil and 1.0 emulsion wax to 85 C.
  • DNA and conservants are added to the cream mass under certain conditions (temperature, pH of medium, adding sequence).
  • the resulting cream is a homogeneous emulsion of white or light-yellow color with a pleasant odor.
  • the pharmacological investigations of the produced cream have shown that it possesses antioxidating, photoprotective, hydrate, proliferative and antiinflammatory properties. No allergic complications and irritating effect were observed. Cream penetrates efficiently through the skin barrier.
  • Lotion is a gel-like liquid with a pleasant odor.
  • Experimental clinical investigations of the produced lotion have shown that it features an antiseborrheal photoprotective and nutrient effect. No side effects and allergenic action was discovered.
  • a container is loaded with 35.0 (w%) ethyl alcohol, 5.0 glycerine, distilled water, 0.25 DNA, 0.005 food dyestuffs (indigocarmine blue and yellow tartrasine) and 1.0 of aromatizer. All ingredients are mixed.
  • the resulting dentifrice water is a bluish liquid with pleasant odor and taste.
  • Clinical investigations have disclosed its antiinflammatory, wound-healing and antiseptic effect without any allergic and other complications.
  • the fatty and aqueous phases are prepared.
  • aqueous phase 3.0 (w%) glycerine or 1.2 propylene and 0.15 triethanolamine are heated to 75-80 C.
  • the fatty phase is prepared by melting to 80-85 C 1.0 anhydrous lanolin, 1.0 distilled monoglycerides or glycerine monostearate or DEG stearate, 2.0 cosmetic stearine, 2.0 perfumery oil or butylstearate or isopropylpalmitate or propylmiristate, 1.0 bee wax, 2.0 of mixture of mono- and diesters, polyethylene glycol-400 and stearate.
  • the fatty and aqueous phases are mixed and doped with 3.33 conservants, 0.25 sodium salt of DNA, and 0.4 aromatizer.
  • the resulting cream is a homogeneous thick white mass with a pleasant smell.
  • the pharmacological investigations of the obtained cream have exhibited the presence of hydrate ability, photoprotective, antioxidating and antiinflammatory properties.
  • the aqueous phase is made by taking 3.0 (w%) glycerine or 1.2 propyleneglycol, 0.2 copolymer of acrylic acid and polyallyl ethers of pentaerythritol or sodium salt of copolymer of acrylic acid and heating them to 75-80 C.
  • the fatty phase is made by taking 0.5 oxyethylated lanolin, 1.0 distilled monoglycerides or glycerine monostearate or stearate DEG, 1.0 bee wax, 3.0 perfumery oil or butylstearate or isopropylpalminate, 3.0 of a mixture of mono- and diesters of polyethyleneglycol-400 and stearine, 2.0 of a mixture of mono- and diesters of oleic acid and polyethyleneglycol-400, 0.5 of alcohols, fraction C 17 -C 18 and heating them to 80-85 C. Then the aqueous and fatty phases are mixed and doped with 3.33 complex conservant, 0.25 sodium salt of DNA and 0.2 aromatizer.
  • the resulting cream is a homogeneous white or light-yellow emulsion with a pleasant odor.
  • the pharmacological investigations of the produced cream have revealed the presence of its antioxidating, photoprotective, hydrating, proliferating and antiinflammatory properties. There were no allergic complications and irritating effect.
  • the 'cream penetrates efficiently through the skin barrier.

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Claims (18)

  1. Verbindung der niedermolekularen DNA, welche aus Störmilch hergestellt ist, mindestens 80 Gew,-% eines Natriumsalz von DNA, nicht mehr als 1,5 Gew.-% an Proteinen und nicht mehr als 17 Gew.-% an Feuchtigkeit enthält, wobei das Natriumsalz von DNA ohne eine wesentliche Menge an Polysacchariden ist, ein Molekulargewicht von nicht mehr als 12,0 MD besitzt und einen hyperchromen Effekt von nicht weniger als 37% besitzt.
  2. Verbindung nach Anspruch 1, dadurch gekennzeichnet, daß die DNA mit mehrwertigen Metallen kombiniert ist.
  3. Verbindung nach Anspruch 2, dadurch gekennzeichnet, daß die Metalle aus der Zn, Co, Ni, Fe, Pd, Pt einschließenden Gruppe ausgewählt sind.
  4. Pharmazeutisches Präparat, welches eine wirksame Menge eines aktiven Grundbestandteils in der Form von niedermolekularer DNA aus Störmilch gemäß Anspruch 1 in Kombination mit einem pharmazeutisch akzeptablen Additiv, Verdünnungsmitteln oder Füllstoffen enthält.
  5. Pharmazeutisches Präparat nach Anspruch 4, dadurch gekennzeichnet, daß die niedermolekulare DNA aus Störmilch mit mehrwertigen Metallen kombiniert ist.
  6. Pharmazeutisches Präparat nach Anspruch 4 oder 5, dadurch gekennzeichnet, daß es die Form einer Lösung besitzt.
  7. Pharmazeutisches Präparat nach Anspruch 6, dadurch gekennzeichnet, daß es in der Form von Injektionen verabreichbar ist.
  8. Pharmazeutisches Präparat nach Anspruch 6, dadurch gekennzeichnet, daß es für eine äußere Anwendung gedacht ist.
  9. Pharmazeutisches Präparat nach Anspruch 7, dadurch gekennzeichnet, daß dessen bevorzugter DNA-Gehalt von 0,5 bis 2,5% reicht.
  10. Pharmazeutisches Präparat nach Anspruch 8, dadurch gekennzeichnet, daß dessen bevorzugter Gehalt der DNA von 0,2 bis 2% reicht.
  11. Pharmazeutisches Präparat nach Anspruch 5, dadurch gekennzeichnet, daß das Verhältnis von DNA und mehrwertigem Metall von 1:1 bis 1:1000 reicht.
  12. Verbindung nach Anspruch 1, dadurch gekennzeichnet, daß sie in der Form eines kosmetischen Produkts in Kombination mit pharmazeutisch akzeptablen Trägern, Füllstoffen, Additiven verwendet wird.
  13. Verbindung nach Anspruch 12, dadurch gekennzeichnet, daß sie ein Zahnputzwasser oder eine Zahnpasta ist.
  14. Verbindung nach Anspruch 12, dadurch gekennzeichnet, daß sie eine Lotion ist.
  15. Verbindung nach Anspruch 12, dadurch gekennzeichnet, daß sie die Form einer Creme oder eines Gels besitzt.
  16. Verfahren zur Extraktion von niedermolekularer DNA aus Störmilch gemäß Anspruch 1, welches beinhaltet ein Homogenisieren von gefrorener Milch in einem Citratsalzpuffer, Deproteinisieren von DNA mittels Detergentien, Denaturieren und Separieren von Protein, anschließendes Abbauen von DNA, dessen Separieren von spezifischen Zusätzen und Separieren des Zielproduktes, dadurch gekennzeichnet, daß diesem Abbau von DNA die Elektrodialyse und/oder Ultrafiltrationsstufen vorangehen und das Zielprodukt mit Ethylalkohol in der Form eines Pulvers aus Salzlösungen ausgefällt wird.
  17. Verfahren nach Anspruch 16, dadurch gekennzeichnet, daß eine Ultrafiltrationskonzentrierung bei einer Temperatur von 20-24°C und einem Druck von 2-3 kg/cm2 durchgeführt wird.
  18. Verfahren nach Anspruch 16, dadurch gekennzeichnet, daß eine Elektrodialyse bei einer Temperatur von 15-20°C und einer Stromdichte von 1,0-1,5 A/dm2 durchgeführt wird.
EP94913856A 1993-05-24 1994-04-14 Niedermolekulare dna aus störmilch, methode zur gewinnung der dna und ein pharmazeutisches präparat darauf basierend Expired - Lifetime EP0712936B1 (de)

Applications Claiming Priority (17)

Application Number Priority Date Filing Date Title
RU93020154A RU2043767C1 (ru) 1993-05-24 1993-05-24 Способ подавления вич-инфекции
RU93020154 1993-05-24
RU93020155 1993-05-24
RU93020155/14A RU2034554C1 (ru) 1993-05-24 1993-05-24 Способ выделения днк из молок осетровых рыб (его варианты)
RU93026472/14A RU2049461C1 (ru) 1993-06-10 1993-06-10 Биологически активная добавка для косметических средств
RU93026472 1993-06-10
RU93046328 1993-10-11
RU93046329/14A RU2078581C1 (ru) 1993-10-11 1993-10-11 Способ стимуляции лейкопоэза
RU93046327 1993-10-11
RU93046328/14A RU2094438C1 (ru) 1993-10-11 1993-10-11 Инъекционное средство для гемокоррекции
RU93046326A RU2071780C1 (ru) 1993-10-11 1993-10-11 Способ лечения медикаментозных эзофаго-гингиво-стоматитов
RU93046327/14A RU93046327A (ru) 1993-10-11 Гемокорректор
RU93046329 1993-10-11
RU93046326 1993-10-11
RU93051600 1993-11-19
RU93051600/14A RU93051600A (ru) 1993-11-19 Противомикробное и противовирусное средство
PCT/RU1994/000084 WO1994028153A2 (fr) 1993-05-24 1994-04-14 Adn de faible masse moleculaire tiree de laitance d'esturgeon, procede d'obtention de l'adn et preparation pharmaceutique a base de celui-ci

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EP0712936A1 EP0712936A1 (de) 1996-05-22
EP0712936A4 EP0712936A4 (de) 1996-10-25
EP0712936B1 true EP0712936B1 (de) 2002-02-06

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WO1996009832A1 (fr) * 1994-09-29 1996-04-04 Nauchno-Proizvodstvennoe Predpriyatie 'farmek' Hybride d'adn-arn et preparation d'adn, procedes d'obtention dudit hybride et de ladite preparation a partir de laitance d'esturgeon, et compose pharmaceutique a base dudit hybride d'adn-arn
JP3366769B2 (ja) * 1995-03-03 2003-01-14 雪印乳業株式会社 白子を配合した栄養組成物
RU2172632C1 (ru) * 2000-12-01 2001-08-27 Каплина Элли Николаевна Способ получения иммунотропного противовирусного препарата
KR100475255B1 (ko) * 2002-03-18 2005-03-10 권구상 캐비어 추출물을 포함하는 나노좀의 유액 조성물 및 그를포함하는 화장료 조성물
DE10241938A1 (de) * 2002-09-10 2004-03-25 Noxxon Pharma Ag Verfahren zur Selektion von Nukleinsäureliganden
EP1908455A4 (de) * 2005-06-13 2013-12-04 Nissei Bio Co Ltd Haarpflege-zubereitung
WO2018202028A1 (zh) * 2017-05-04 2018-11-08 高雄医学大学 一种组合物用于制备促进个体毛发生长、预防毛发脱落或促进个体毛囊干细胞生成的药物的用途
IT201900003009A1 (it) * 2019-03-01 2020-09-01 Curasept A D S S R L Prodotto per l’igiene orale
JP7015064B2 (ja) * 2019-08-05 2022-02-02 日生バイオ株式会社 角栓除去剤
CN117598964B (zh) * 2023-11-16 2024-04-30 广州中妆美业化妆品有限公司 一种具有修护、抗氧化和抗皱紧致功效的茶与鱼子酱组合物及其应用

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FR2536278A1 (fr) * 1982-11-18 1984-05-25 Dupont Michele Nouvelle composition therapeutique utile notamment pour la cicatrisation des plaies
US4885157A (en) * 1987-02-13 1989-12-05 Fiaschetti Mary G Dermal cosmetic composition and applications therefor
CA1297431C (en) * 1987-04-24 1992-03-17 F. Hoffmann-La Roche Ag Process for the isolation of nucleic acids
IT1222701B (it) * 1987-09-23 1990-09-12 Crinos Industria Farmaco Composizione ad uso topico avente attivita' tricogena,antiforfora ed antiseborroica
US4923978A (en) * 1987-12-28 1990-05-08 E. I. Du Pont De Nemours & Company Process for purifying nucleic acids
US5010183A (en) * 1989-07-07 1991-04-23 Macfarlane Donald E Process for purifying DNA and RNA using cationic detergents
WO1994004551A1 (en) * 1992-08-26 1994-03-03 Jury Petrovich Vainberg Method of obtaining sodium salt of native dna
US5547684A (en) * 1993-02-10 1996-08-20 Pharmec Company Cosmetic composition containing a DNA-sodium salt and methods of making and using the same

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JPH08509000A (ja) 1996-09-24
EP0712936A1 (de) 1996-05-22
AU6584994A (en) 1994-12-20
EP0712936A4 (de) 1996-10-25
WO1994028153A3 (fr) 1995-03-30
WO1994028153A2 (fr) 1994-12-08
ES2171450T3 (es) 2002-09-16
ATE212848T1 (de) 2002-02-15
DE69429812D1 (de) 2002-03-21
DE69429812T2 (de) 2002-08-14

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